Antibiotic-resistant bacteria represent a growing global problem, as these bacteria cannot be killed or made to stop dividing by antibiotics. The generation time of bacteria can in many cases be very fast (around 20 minutes), and due to the short generation time and relative genetic instability of bacteria, the bacteria may quickly acquire resistance towards antibiotics. There is an increasing prevalence of antibiotic-resistant bacterial infections in the human population, and some of these bacteria have even become multi-resistant, sometimes meaning that there are no efficient antibiotics available to halt their growth. These multi-resistant bacteria are a serious public health problem as patients infected with such bacteria may die due to that their bacterial infections cannot be treated.
The traditional approaches for the identification and study of microorganisms, including but not limited to bacteria, fungi, parasites and viruses, and their resistance to antibiotics that kill or inhibit the growth of the microorganism, have mainly in the example of bacteria been limited to Luria broth (LB) agar plates. These LB agar plates have then been made to contain bactericidal and bacteriostatic antibiotics of defined concentrations. Such two-dimensional (2D) culture methods for antibiotic susceptibility testing (AST) and evaluation of the effects of antibiotics or other test substances on microorganisms have several limitations. For instance, these setups normally require that the microorganisms, e.g. bacteria, are cultured over night to allow for a clear result readout showing if a particular bacteria strain is resistant or not to a given antibiotic. In addition, generally only a single antibiotic concentration can be tested per LB agar plate.
Another prior art AST approach uses a so-called E-test. The E-test is basically an agar diffusion method and uses a rectangular strip impregnated with different concentrations of a test substance to be evaluated for its effect as an antibiotic. In a typical approach, bacteria are spread and grown in a 2D culture on top of an agar plate, where after the E-test strip is placed on top of the agar plate. The E-test strip releases the test substance by diffusion and the growth inhibitory effects of the released test substance are typically inspected after 24 hours of incubation. A limitation of this approach is, in addition to the very long incubation time, that readouts of the inhibitory concentration of the test substance is only possible in distinct digital steps and in the selected concentrations used in the E-test strip.
A further traditional AST approach uses a microtiter plate assay with different concentrations of a test substance in different wells. The microtiter plate with added bacteria is usually incubated overnight and the inhibitory effects on the bacteria are evaluated by measuring the optical turbidity in the different wells. This approach has basically the same shortcomings as when using E-test strips.
In order to reduce the AST time, microfluidic channel systems for rapid AST (RAST) have been developed. Such RAST approaches include droplet-based microfluidic channel systems in which bacteria are captured in a droplet that includes an antibiotic [1-3]. A limitation with the droplet-based system is that only a single antibiotic concentration can be tested. Other RAST approaches include using gas permeable polydimethylsiloxane (PDMS) microchannels [4], dielectrophoretic capturing of bacteria in microfluidic electrode structures [5-6], preloaded PDMS layers with antibiotics [7], covalently binding bacteria to microfluidic channels and subjecting them to mechanical shear stress [8], using asynchronous magnetic bead rotation (AMBR) biosensors [9] or tracking single cell growth in a microfluidic agarose channel system [10]. A major limitation of these various RAST approaches is that they can only test a single antibiotic concentration or a set of a few selected antibiotic concentrations.
It has further been proposed to use a microfluidic system for analysis of antibiotic susceptibility of bacterial biofilms [11]. Their microfluidic system, however, requires 24 hours of incubation and that the bacteria to be tested contain a plasmid able to express green fluorescent protein (GFP).
Hence, there is still a need for fast methods and systems for response testing of microorganism that do not have the disadvantages of the prior art.